1. tRNA and rRNA processing1. tRNAs and rRNAs of both eukaryotes and prokaryotesare oftensynthesized as longer precursors, and need to be processed tobecome the final products.2. rRNA and tRNAs are processed by intron splicing, cleavage (cut inthe middle without religation), and trimming (cut from ends). SomerRNA/tRNA precursors can be cut to produce several rRNA as wellas tRNAs. Many different RNases are involved.

2. RibozymeAn E. coli RNase called RNase P isinvolved in tRNA processing.RNase P is composed of a RNA calledM1 RNA and a 14kD protein.It was found in 1983 by Thomas Cech(who also first found self-splicing in1982) that M1 RNA cuts the pre-RNA(Pre-tRNAtyr).The M1 RNA and other catalytic RNAswere later named as ribozymes(RNAenzymes).

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Ribozyme catalysisResult:(1) pTyr (pre-tRNATyr) was cutby M1 RNA, but P4.5 (a pre-rRNA) was not cut,demonstrating specificity(2) M1 need more Mg than theRNase P holoenzyme, indicatingthat although RNA alone iscatalytically active, the proteincomponent changes theproperties of the enzyme.Experiment:32P-label pre-tRNA (and a control pre-rRNA),incubate with M1 RNA,run a gel to see if pre-tRNA is cut by M1.M1+PM1

Self-cleavage ribozymes(a)Secondary structure of the HDV (Hepatitis delta virus)ribozyme;the cytosine (C75) is essential to catalysis. The 5'endof the HDVribozyme RNA marks the cleavage site (between U-C).(b)3D structure of the HDV ribozyme. The C75folds to become closeto the cleavage site and the 5'end of the ribozyme.(c) Chemistry of the catalysis by C75, in which the protonated C75donates a proton to catalyze the cleave. (Nature2002, 418:222)

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3. Trans-splicingThe pre-mRNA splicing discussed before involved cut/religation offragments within the same RNA molecule, it is called cis-splicing.It was found that, in worm trypanosomes, fragments of differentpre-mRNAs can be spliced together, it is called trans-splicing.Trans-splicing involves transesterificationreaction without lariat.The previously discussed pre-tRNA cleavage by M1 RNA of RNaseP of E. coli can also be called trans-splicing (without re-ligation).(RNA1) (RNA2)